CN101020051A - Prepn and usage of outer membrane protein subunit vaccine of seawater fish morbid vibrio - Google Patents
Prepn and usage of outer membrane protein subunit vaccine of seawater fish morbid vibrio Download PDFInfo
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- CN101020051A CN101020051A CNA200610124342XA CN200610124342A CN101020051A CN 101020051 A CN101020051 A CN 101020051A CN A200610124342X A CNA200610124342X A CN A200610124342XA CN 200610124342 A CN200610124342 A CN 200610124342A CN 101020051 A CN101020051 A CN 101020051A
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Abstract
The present invention is preparation and usage of outer membrane protein subunit morbid vibrio vaccine for seawater fish. The vaccine is prepared through extracting two or more of Vibrio alginolyticus, Vibrio Hrveyi, Vibrio vulnificus, Vibrio parahaemolyticus, etc, and adding immunostimulating complex. The vaccine preparing process includes culturing vibrio, extracting outer membrane protein and preparing vaccine. The vaccine containing partial pathogen and no infecting component has stable immunological specificity and no hidden danger of restoring toxici. The vaccine is used in preventing fishes' skin ulcer, eyeball turbidity and other vibrio caused diseases and may be used widely for immunizing seawater fish.
Description
Technical field:
The invention belongs to a kind of preparation and using method of outer membrane protein subunit vaccine of beach vibrio piscium disease.
Background technology:
In recent years, along with developing rapidly of mariculture industry, marine fish cage culture scale also constantly enlarges, because culturing sea area net cage quantity increases, cultivation density strengthens, the breed water environment runs down, the generation of disease is also more and more frequent, that bacterial disease becomes is the most common in the aquaculture, endanger one of severe diseases, often cause the mortality of cultured fishes, wherein vibriosis (Vibrosis) is again a disease the most common in the marine fish culture, that harm is maximum, already causes enormous economic loss for marine fish culture.
For a long time, tradition is prevented and treated method and generally is to use chemicals and antibiotic, uses these medicines that pathogen is developed immunity to drugs in a large number, destroys the microecosystem of breeding water body, also can cause medicine intravital residual, have a strong impact on the quality of cultivated animals at cultivated animals.Therefore, in existing technology, have only the production and the application of Vibrio anguillarum thalline vaccine and vibrio mimicus coupled subunit vaccine at present.Vibrio anguillarum thalline vaccine because have that virulence rebounds easily, that using dosage is big, immunne response level body is lower etc. is former thereby cause immune effect not good.The vibrio mimicus coupled subunit vaccine has more single-minded anti-vibrio specificity in aquaculture production, relatively poor to the pathogenic microbial disease defense reaction of other vibrios, has very significantly limitation in production application.
Summary of the invention
The objective of the invention is provides a kind of preparation and using method of outer membrane protein subunit vaccine of beach vibrio piscium disease in order to remedy above-mentioned the deficiencies in the prior art.
The technical scheme taked of the present invention is for achieving the above object:
The preparation method of outer membrane protein subunit vaccine of seawater fish morbid vibrio, be suitable for and make the vaccine that multiple seawater fish improves the anti-vibrio of cultured output, used strain is: the strain of two or more in vibrio alginolyticus, vibrio harveyi, Vibrio vulnificus, Vibrio anguillarum, vibrio parahaemolytious, vibrio mimicus and the vibrio fluvialis; This method is: the preparation of the cultivation of antibacterial, the extraction of outer membrane protein, vaccine.
The preparation method of this outer membrane protein subunit vaccine of seawater fish morbid vibrio, according to the following steps:
The cultivation of antibacterial: antibacterial is adopted tryptone soya peptone fluid medium TSB activation earlier, under 28 ℃, 150r/min shaken cultivation 18h gets 1mL bacterium liquid then and coats that to contain 2%NaCl and initial pH be 7.2 tryptone soya peptone Agar Plating TSA, leaves standstill under 28 ℃ and cultivates 18h;
The extraction of outer membrane protein Omp: with pH is that 7.2 0.01mol/L PBS washes bacterium colony, 4 ℃, the centrifugal 10min of 7000r/min, repeat to wash 2 times, getting bacterium mud suspends with PBS, ultrasonic disruption 7min under the condition of ice bath, 4 ℃, the centrifugal 15min of 7000r/min, get supernatant and remove big bacterial debris, 4 ℃, the centrifugal 1h of 35000r/min, precipitation is dissolved in sarcosyl, 37 ℃ the effect 60min after 4 ℃, the centrifugal 1h of 35000r/min is the outer membrane protein extract with above-mentioned PBS collecting precipitation ,-20 ℃ of preservations;
The preparation of vaccine: with protein: cholesterol+lecithin: the ratio of adjuvant=1: 1: 5, wherein adjuvant is Quil-A, prepare outer membrane protein subunit vaccine ISCOM-Omp as follows: in 1mgOmp, add 100 μ L 10mg/mL with the cholesterol of 20%OG preparation and the mixture of lecithin, wherein the ratio of cholesterol and lecithin is 1: 1, add 5 μ L 100mg/mL Quil-A again, with the buffer adjusted volume to 1mL, mixing on the vortex instrument, place ultrasonic 5min on the ultrasonic disruption instrument again, use the 0.01mol/L PBS of pH7.2 to dialyse after being placed on room temperature 2h, move into 4 ℃ of refrigerators behind the 6h, dialysed 3 days, change dialysis solution 3-4 time every day.
The using method of this outer membrane protein subunit vaccine of seawater fish morbid vibrio:
(1) infusion method: should carry out immersion immunity during the seedling.Method is put in the container that fills vaccine for the fish that will need immunity, and soak time is one day;
(2) injection: the juvenile fish stage can be carried out the lumbar injection immunity, and injected dose is the 0.1ml/ tail:
(3) oral immunity: the juvenile fish stage also can be carried out oral vaccine, and every double centner fish 0.05ml every day mixes to raise and throws something and feeds, and connects to feed 3 days.
Advantage of the present invention and effect:
1. the immune effect of multicomponent vaccine is better than the one-component vaccine;
2. a part that contains pathogen in the vibrio vaccine, thereby this vaccine is more definite on chemical property, immunologic opsonin is more stable;
3. outer membrane protein subunit vaccine of seawater fish morbid vibrio highly effective and safe, drug residue free pollutes;
4. infect composition and be removed, relevant residual toxicity or the regressive hidden danger of toxicity no longer exist:
5. the outer membrane protein subunit vaccine of seawater fish morbid vibrio wide spectrum is resisted the invasion and attack infection of various seawater fish morbid vibrios, and its applicable object is various marine fishs:
6. easy to use, can the multipath immunity inoculation, can during seedling, carry out immersion immunity, can carry out injecting immune in the juvenile fish stage again, also can carry out oral immunity.
7. this vaccine immunity protective rate 85-90%, specifically protect effect to see the following form:
The trial effect of table 1 vaccine immersion immunity in the cage culture red snapper
| The place | Group | Experimental period (moon) | Experiment fish (tail) | Dead fish (tail) | Mortality rate (%) | Survival rate (%) |
| Zhanjiang, Leizhou | Immune group matched group immune group matched group | 9 9 9 9 | 500 500 500 500 | 253 53 224 | 14.4 50.6 10.6 44.8 | 85.6 49.4 89.4 55.2 |
The trial effect of table 2 vaccine injecting immune in the cage culture cabrilla
| The place | Group | Experimental period (moon) | Experiment fish (tail) | Dead fish (tail) | Mortality rate (%) | Survival rate (%) |
| Hainan, Yangjiang | Immune group matched group immune group matched group | 9 9 9 9 | 500 500 500 500 | 47 238 56 257 | 9.4 47.6 11.3 51.4 | 90.6 52.4 88.7 48.6 |
The trial effect of table 3 vaccine oral immunity in the cage culture rock salmon
| The place | Group | Experimental period (moon) | Experiment fish (tail) | Dead fish (tail) | Mortality rate (%) | Survival rate (%) |
| The spy is the island | The immune group matched group | 9 9 | 500 500 | 67 248 | 13.4 49.6 | 86.6 50.4 |
| The Sanya | The immune group matched group | 9 9 | 500 500 | 52 228 | 10.4 54.4 | 89.6 45.6 |
The specific embodiment
The present invention further specifies with the following example.
Embodiment 1
Respectively vibrio alginolyticus and vibrio harveyi are adopted tryptone soya peptone fluid medium (TSB) activation earlier, under 28 ℃, 150r/min shaken cultivation 18h, getting 1mL bacterium liquid separate application then is 7.2 tryptone soya peptone Agar Plating (TSA) in containing 2%NaCl and initial pH, leaves standstill under 28 ℃ and cultivates 18h.Then, extract vibrio alginolyticus and vibrio harveyi outer membrane protein respectively.Method is: with pH is that 7.2 0.01mol/L PBS washes bacterium colony respectively, 4 ℃, and the centrifugal 10min of 7000r/min, repeat to wash 2 times, get bacterium mud and suspend, ultrasonic disruption 7min under the condition of ice bath with PBS, 4 ℃, the centrifugal 15min of 7000r/min gets supernatant and removes big bacterial debris, 4 ℃, the centrifugal 1h of 35000r/min, precipitation is dissolved in sarcosyl, 37 ℃ the effect 60min after 4 ℃, the centrifugal 1h of 35000r/min is the outer membrane protein extract with above-mentioned PBS collecting precipitation ,-20 ℃ of preservations.Then prepare vibrio alginolyticus and vibrio harveyi outer membrane protein subunit vaccine respectively.Method is: with protein: (cholesterol+lecithin): the ratio of adjuvant (Quil-A)=1: 1: 5, prepare ISCOM-Omp as follows: the Omp that gets 1mg vibrio alginolyticus and vibrio harveyi adds 100 μ L 10mg/mL respectively with the cholesterol of 20%OG preparation and the mixture (ratio of cholesterol and lecithin is 1: 1) of lecithin, add 5 μ L100mg/mL Quil-A again, with the buffer adjusted volume to 1mL, mixing on the vortex instrument, place ultrasonic 5min on the ultrasonic disruption instrument again, use the 0.01mol/L PBS of pH7.2 to dialyse after being placed on room temperature 2h, move into 4 ℃ of refrigerators behind the 6h, dialysed 3 days, and changed dialysis solution 3-4 time every day.At last the vibrio alginolyticus and the vibrio harveyi outer membrane protein subunit vaccine equal proportion that make are mixed, obtain the morbid vibrio outer membrane protein subunit vaccine.
Embodiment 2
Respectively vibrio alginolyticus, vibrio harveyi, Vibrio vulnificus and Vibrio anguillarum are adopted tryptone soya peptone fluid medium (TSB) activation earlier, under 28 ℃, 150r/min shaken cultivation 18h, getting 1mL bacterium liquid separate application then is 7.2 tryptone soya peptone Agar Plating (TSA) in containing 2%NaCl and initial pH, leaves standstill under 28 ℃ and cultivates 18h.Then, extract vibrio alginolyticus, vibrio harveyi, Vibrio vulnificus and Vibrio anguillarum outer membrane protein respectively.Method is: with pH is that 7.2 0.01mol/L PBS washes bacterium colony respectively, 4 ℃, and the centrifugal 10min of 7000r/min, repeat to wash 2 times, get bacterium mud and suspend, ultrasonic disruption 7min under the condition of ice bath with PBS, 4 ℃, the centrifugal 15min of 7000r/min gets supernatant and removes big bacterial debris, 4 ℃, the centrifugal 1h of 35000r/min, precipitation is dissolved in sarcosyl, 37 ℃ the effect 60min after 4 ℃, the centrifugal 1h of 35000r/min is the outer membrane protein extract with above-mentioned PBS collecting precipitation ,-20 ℃ of preservations.Then prepare vibrio alginolyticus, vibrio harveyi, Vibrio vulnificus and Vibrio anguillarum outer membrane protein subunit vaccine respectively.Method is: with protein: (cholesterol+lecithin): the ratio of adjuvant (Quil-A)=1: 1: 5, prepare ISCOM-Omp as follows: get the 1mg vibrio alginolyticus, vibrio harveyi, the Omp of Vibrio vulnificus and Vibrio anguillarum adds 100 μ L 10mg/mL respectively with the cholesterol of 20%OG preparation and the mixture (ratio of cholesterol and lecithin is 1: 1) of lecithin, add 5 μ L 100mg/mL Quil-A again, with the buffer adjusted volume to 1mL, mixing on the vortex instrument, place ultrasonic 5min on the ultrasonic disruption instrument again, use the 0.01mol/L PBS of pH7.2 to dialyse after being placed on room temperature 2h, move into 4 ℃ of refrigerators behind the 6h, dialysed 3 days, and changed dialysis solution 3-4 time every day.At last the vibrio alginolyticus, vibrio harveyi, Vibrio vulnificus and the Vibrio anguillarum outer membrane protein subunit vaccine equal proportion that make are mixed, obtain the morbid vibrio outer membrane protein subunit vaccine.
Embodiment 3
Respectively vibrio alginolyticus, vibrio harveyi, Vibrio vulnificus, Vibrio anguillarum, vibrio parahaemolytious and vibrio mimicus are adopted tryptone soya peptone fluid medium (TSB) activation earlier, under 28 ℃, 150r/min shaken cultivation 18h, getting 1mL bacterium liquid separate application then is 7.2 tryptone soya peptone Agar Plating (TSA) in containing 2%NaCl and initial pH, leaves standstill under 28 ℃ and cultivates 18h.Then, extract vibrio alginolyticus, vibrio harveyi, Vibrio vulnificus, Vibrio anguillarum, vibrio parahaemolytious and vibrio mimicus outer membrane protein respectively.Method is: with pH is that 7.2 0.01mol/L PBS washes bacterium colony respectively, 4 ℃, and the centrifugal 10min of 7000r/min, repeat to wash 2 times, get bacterium mud and suspend, ultrasonic disruption 7min under the condition of ice bath with PBS, 4 ℃, the centrifugal 15min of 7000r/min gets supernatant and removes big bacterial debris, 4 ℃, the centrifugal 1h of 35000r/min, precipitation is dissolved in sarcosyl, 37 ℃ the effect 60min after 4 ℃, the centrifugal 1h of 35000r/min is the outer membrane protein extract with above-mentioned PBS collecting precipitation ,-20 ℃ of preservations.Then prepare vibrio alginolyticus, vibrio harveyi, Vibrio vulnificus, Vibrio anguillarum, vibrio parahaemolytious and vibrio mimicus outer membrane protein subunit vaccine respectively.Method is: with protein: (cholesterol+lecithin): the ratio of adjuvant (Quil-A)=1: 1: 5, prepare ISCOM-Omp as follows: get the 1mg vibrio alginolyticus, vibrio harveyi, Vibrio vulnificus, Vibrio anguillarum, the Omp of vibrio parahaemolytious and vibrio mimicus adds 100 μ L10mg/mL respectively with the cholesterol of 20%OG preparation and the mixture (ratio of cholesterol and lecithin is 1: 1) of lecithin, add 5 μ L 100mg/mL Quil-A again, with the buffer adjusted volume to 1mL, mixing on the vortex instrument, place ultrasonic 5min on the ultrasonic disruption instrument again, use the 0.01mol/L PBS of pH7.2 to dialyse after being placed on room temperature 2h, move into 4 ℃ of refrigerators behind the 6h, dialysed 3 days, and changed dialysis solution 3-4 time every day.At last the vibrio alginolyticus, vibrio harveyi, Vibrio vulnificus, Vibrio anguillarum, vibrio parahaemolytious and the vibrio mimicus outer membrane protein subunit vaccine equal proportion that make are mixed, obtain the morbid vibrio outer membrane protein subunit vaccine.
Embodiment 4
Respectively vibrio alginolyticus, vibrio harveyi and Vibrio vulnificus are adopted tryptone soya peptone fluid medium (TSB) activation earlier, under 28 ℃, 150r/min shaken cultivation 18h, getting 1mL bacterium liquid separate application then is 7.2 tryptone soya peptone Agar Plating (TSA) in containing 2%NaCl and initial pH, leaves standstill under 28 ℃ and cultivates 18h.Then, extract vibrio alginolyticus, vibrio harveyi and Vibrio vulnificus outer membrane protein respectively.Method is: with pH is that 7.2 0.01mol/L PBS washes bacterium colony respectively, 4 ℃, and the centrifugal 10min of 7000r/min, repeat to wash 2 times, get bacterium mud and suspend, ultrasonic disruption 7min under the condition of ice bath with PBS, 4 ℃, the centrifugal 15min of 7000r/min gets supernatant and removes big bacterial debris, 4 ℃, the centrifugal 1h of 35000r/min, precipitation is dissolved in sarcosyl, 37 ℃ the effect 60min after 4 ℃, the centrifugal 1h of 35000r/min is the outer membrane protein extract with above-mentioned PBS collecting precipitation ,-20 ℃ of preservations.Then prepare vibrio alginolyticus, vibrio harveyi and Vibrio vulnificus outer membrane protein subunit vaccine respectively.Method is: with protein: (cholesterol+lecithin): the ratio of adjuvant (Quil-A)=1: 1: 5, prepare ISCOM-Omp as follows: get the 1mg vibrio alginolyticus, vibrio harveyi, the Omp of Vibrio vulnificus adds 100 μ L 10mg/mL respectively with the cholesterol of 20%OG preparation and the mixture (ratio of cholesterol and lecithin is 1: 1) of lecithin, add 5 μ L 100mg/mL Quil-A again, with the buffer adjusted volume to 1mL, mixing on the vortex instrument, place ultrasonic 5min on the ultrasonic disruption instrument again, use the 0.01mol/L PBS of pH7.2 to dialyse after being placed on room temperature 2h, move into 4 ℃ of refrigerators behind the 6h, dialysed 3 days, and changed dialysis solution 3-4 time every day.At last the vibrio alginolyticus, vibrio harveyi and the Vibrio vulnificus outer membrane protein subunit vaccine equal proportion that make are mixed, obtain the morbid vibrio outer membrane protein subunit vaccine.
Embodiment 5
Respectively vibrio alginolyticus, vibrio harveyi, Vibrio anguillarum and vibrio parahaemolytious are adopted tryptone soya peptone fluid medium (TSB) activation earlier, under 28 ℃, 150r/min shaken cultivation 18h, getting 1mL bacterium liquid separate application then is 7.2 tryptone soya peptone Agar Plating (TSA) in containing 2%NaCl and initial pH, leaves standstill under 28 ℃ and cultivates 18h.Then, extract vibrio alginolyticus, vibrio harveyi, Vibrio vulnificus, Vibrio anguillarum and vibrio parahaemolyticus outer membrane protein respectively.Method is: with pH is that 7.2 0.01mol/LPBS washes bacterium colony respectively, 4 ℃, and the centrifugal 10min of 7000r/min, repeat to wash 2 times, get bacterium mud and suspend, ultrasonic disruption 7min under the condition of ice bath with PBS, 4 ℃, the centrifugal 15min of 7000r/min gets supernatant and removes big bacterial debris, 4 ℃, the centrifugal 1h of 35000r/min, precipitation is dissolved in sarcosyl, 37 ℃ the effect 60min after 4 ℃, the centrifugal 1h of 35000r/min is the outer membrane protein extract with above-mentioned PBS collecting precipitation ,-20 ℃ of preservations.Then prepare vibrio alginolyticus, vibrio harveyi, Vibrio vulnificus, Vibrio anguillarum, vibrio parahaemolyticus outer membrane protein subunit vaccine respectively.Method is: with protein: (cholesterol+lecithin): the ratio of adjuvant (Quil-A)=1: 1: 5, prepare ISCOM-Omp as follows: get the 1mg vibrio alginolyticus, vibrio harveyi, Vibrio vulnificus, the Omp of Vibrio anguillarum and vibrio parahaemolytious adds 100 μ L 10mg/mL respectively with the cholesterol of 20%OG preparation and the mixture (ratio of cholesterol and lecithin is 1: 1) of lecithin, add 5 μ L 100mg/mL Quil-A again, with the buffer adjusted volume to 1mL, mixing on the vortex instrument, place ultrasonic 5min on the ultrasonic disruption instrument again, use the 0.01mol/L PBS of pH7.2 to dialyse after being placed on room temperature 2h, move into 4 ℃ of refrigerators behind the 6h, dialysed 3 days, and changed dialysis solution 3-4 time every day.At last the vibrio alginolyticus, vibrio harveyi, Vibrio vulnificus, Vibrio anguillarum and the vibrio parahaemolyticus outer membrane protein subunit vaccine equal proportion that make are mixed, obtain the morbid vibrio outer membrane protein subunit vaccine.
Embodiment 6
Respectively vibrio alginolyticus, vibrio harveyi, Vibrio vulnificus, Vibrio anguillarum, vibrio parahaemolytious, vibrio mimicus and vibrio fluvialis are adopted tryptone soya peptone fluid medium (TSB) activation earlier, under 28 ℃, 150r/min shaken cultivation 18h, getting 1mL bacterium liquid separate application then is 7.2 the old Agar Plating of tryptone Semen sojae atricolor (TSA) in containing 2%NaCl and initial pH, leaves standstill under 28 ℃ and cultivates 18h.Then, extract vibrio alginolyticus, vibrio harveyi, Vibrio vulnificus, Vibrio anguillarum, vibrio parahaemolytious, vibrio mimicus and vibrio fluvialis outer membrane protein respectively.Method is: with pH is that 7.2 0.01mol/L PBS washes bacterium colony respectively, 4 ℃, and the centrifugal 10min of 7000r/min, repeat to wash 2 times, get bacterium mud and suspend, ultrasonic disruption 7min under the condition of ice bath with PBS, 4 ℃, the centrifugal 15min of 7000r/min gets supernatant and removes big bacterial debris, 4 ℃, the centrifugal 1h of 35000r/min, precipitation is dissolved in sarcosyl, 37 ℃ the effect 60min after 4 ℃, the centrifugal 1h of 35000r/min is the outer membrane protein extract with above-mentioned PBS collecting precipitation ,-20 ℃ of preservations.Then prepare vibrio alginolyticus, vibrio harveyi, Vibrio vulnificus, Vibrio anguillarum, vibrio parahaemolytious, vibrio mimicus and vibrio fluvialis outer membrane protein subunit vaccine respectively.Method is: with protein: (cholesterol+lecithin): the ratio of adjuvant (Quil-A)=1: 1: 5, prepare ISCOM-Omp as follows: get the 1mg vibrio alginolyticus, vibrio harveyi, Vibrio vulnificus, Vibrio anguillarum, vibrio parahaemolytious, the Omp of vibrio mimicus and vibrio fluvialis adds 100 μ L 10mg/mL respectively with the cholesterol of 20%OG preparation and the mixture (ratio of cholesterol and lecithin is 1: 1) of lecithin, add 5 μ L 100mg/mL Quil-A again, with the buffer adjusted volume to 1mL, mixing on the vortex instrument, place ultrasonic 5min on the ultrasonic disruption instrument again, use the 0.01mol/L PBS of pH7.2 to dialyse after being placed on room temperature 2h, move into 4 ℃ of refrigerators behind the 6h, dialysed 3 days, and changed dialysis solution 3-4 time every day.At last the vibrio alginolyticus, vibrio harveyi, Vibrio vulnificus, Vibrio anguillarum, vibrio parahaemolytious, vibrio mimicus and the vibrio fluvialis outer membrane protein subunit vaccine equal proportion that make are mixed, obtain the morbid vibrio outer membrane protein subunit vaccine.
Claims (3)
1, a kind of preparation method of outer membrane protein subunit vaccine of seawater fish morbid vibrio, be suitable for and make the vaccine that multiple seawater fish improves the anti-vibrio of cultured output, it is characterized in that used strain is: the strain of two or more in vibrio alginolyticus, vibrio harveyi, Vibrio vulnificus, Vibrio anguillarum, vibrio parahaemolytious, vibrio mimicus and the vibrio fluvialis; Steps of the method are: the preparation of the cultivation of antibacterial, the extraction of outer membrane protein, vaccine.
2,, it is characterized in that according to the following steps according to the preparation method of the described outer membrane protein subunit vaccine of seawater fish morbid vibrio of claim 1:
(1) cultivation of antibacterial: antibacterial is adopted tryptone soya peptone fluid medium TSB activation earlier, under 28 ℃, 150r/min shaken cultivation 18h gets 1mL bacterium liquid then and coats that to contain 2%NaCl and initial pH be 7.2 tryptone soya peptone Agar Plating TSA, leaves standstill under 28 ℃ and cultivates 18h;
(2) extraction of adventitia egg Omp.: with pH is that 7.2 0.01mol/L sterile phosphate buffer PBS washes bacterium colony, 4 ℃, the centrifugal 10min of 7000r/min, repeat to wash 2 times, getting bacterium mud suspends with PBS, ultrasonic disruption 7min under the condition of ice bath, 4 ℃, the centrifugal 15min of 7000r/min, get supernatant and remove big bacterial debris, 4 ℃, the centrifugal 1h of 35000r/min, precipitation is dissolved in sarcosyl, 37 ℃ the effect 60min after 4 ℃, the centrifugal 1h of 35000r/min is the outer membrane protein extract with above-mentioned phosphate buffer PBS collecting precipitation ,-20 ℃ of preservations;
(3) preparation of vaccine: with protein: cholesterol+lecithin: the ratio of adjuvant=1: 1: 5, wherein adjuvant is Quil-A, prepare outer membrane protein subunit vaccine ISCOM-Omp as follows: in 1mg Omp, add 100 μ L 10mg/mL with the cholesterol of 2%OG preparation and the mixture of lecithin, wherein the ratio of cholesterol and lecithin is 1: 1, add 5 μ L 100mg/mL Quil-A again, with the buffer adjusted volume to 1mL, mixing on the vortex instrument, place ultrasonic 5min on the ultrasonic disruption instrument again, use the 0.01mol/L PBS of pH7.2 to dialyse after being placed on room temperature 2h, move into 4 ℃ of refrigerators behind the 6h, dialysed 3 days, change dialysis solution 3-4 time every day.
3, according to the using method of the outer membrane protein subunit vaccine of seawater fish morbid vibrio of claim 1 method preparation, it is characterized in that:
(1) infusion method: should carry out immersion immunity during the seedling, method is put in the container that fills vaccine for the fish that will need immunity, and soak time is one day;
(2) injection: the juvenile fish stage can be carried out the lumbar injection immunity, and injected dose is the 0.1ml/ tail;
(3) oral immunity: the juvenile fish stage also can be carried out oral vaccine, and every double centner fish 0.05ml every day mixes to raise and throws something and feeds, and connects to feed 3 days.
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| CN101195823B (en) * | 2007-11-20 | 2010-06-02 | 华东理工大学 | Novel bacterium surface exhibiting system, method and application of the same |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN101195823B (en) * | 2007-11-20 | 2010-06-02 | 华东理工大学 | Novel bacterium surface exhibiting system, method and application of the same |
| CN101361971B (en) * | 2008-10-10 | 2011-01-26 | 福建省农业科学院生物技术研究所 | Preparation technique of Vibrio vulnificus whole-cell immunity stimulated compound vaccine and use thereof in aquatic animal |
| CN101703768B (en) * | 2009-11-02 | 2011-08-17 | 中山大学 | Preparation method of vibrio parahaemolyticus outer membrane protein VP1061 and application of immunity protective function thereof |
| CN101703767B (en) * | 2009-11-02 | 2012-07-25 | 中山大学 | Preparation method of nibrio parahaemolyticus outer membrane protein VP2850and application of immunity protective function thereof |
| CN103408642A (en) * | 2013-06-19 | 2013-11-27 | 中国科学院海洋研究所 | Vibrio harveyi outer membrane protein and application thereof |
| CN103520737A (en) * | 2013-10-15 | 2014-01-22 | 西北农林科技大学 | Carbon nano tube carrier vaccination-free vaccine as well as preparation method and application thereof in preparation of aquatic immune offspring seed |
| CN103520737B (en) * | 2013-10-15 | 2016-05-25 | 西北农林科技大学 | Carbon nanotube carrier is exempted from vaccinate, preparation method and in the application of preparing in aquatic products immunity seed |
| CN105131087A (en) * | 2015-07-22 | 2015-12-09 | 安徽农业大学 | Vibrio mimicus OmpU adhesin protein binding peptide having adhesion antagonistic activity, polypeptide composition and application thereof |
| CN105566461A (en) * | 2015-12-25 | 2016-05-11 | 中山大学 | Bacterial outer membrane protein ompAs-19 after DNA shuffling and application thereof as an immunomodulator |
| CN113144183A (en) * | 2021-04-07 | 2021-07-23 | 汕头大学 | Broad-spectrum vibrio vaccine containing epitope K7 and preparation and application thereof |
| CN113144183B (en) * | 2021-04-07 | 2023-08-18 | 汕头大学 | Broad-spectrum vibrio vaccine containing epitope K7 and preparation and application thereof |
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